Several different strategies have been used to classify porcine reproductive and respiratory syndrome-type-2 viruses into (hopefully) epidemiologically meaningful groups. Here we discuss RFLPs (Restriction Fragment Length Polymorphisms), one of the most commonly used ways of classifying this virus, highlighting how this classification works and its limitations.
RFLP classification for PRRSV was originally proposed in 1998 as a way of differentiating wild (field) strains from vaccine strains (1) at a point of time when sequencing was challenging and expensive. Originally, three restriction enzymes (MluI, HincII and SacII) were used to fragment or cut the PRRSV ORF5 gene, each binding to a specific pattern within the RNA sequence that may occur at different locations across the ORF5 gene. The location (or locations) where each enzyme binds was determined through a laboratory technique called gel electrophoresis. Various patterns of where these enzymes bind and cut the ORF5 gene were denoted as RFLP cut patterns. Today, viral RNA does not need to be exposed to enzymes or run through gel electrophoresis, as RFLPs can be determined computationally from ORF5 sequences.
While originally created to differentiate wild and vaccine strains, the industry expanded the use of RFLP types to further classify the virus into subtypes and help address additional epidemiological questions. For example, are two wild-type viruses the same? Do they behave in similar ways? Are they related evolutionarily? Those questions began being addressed by looking at the RFLP pattern of the virus, but the answers were at best unclear.
One of the issues with RFLP typing is that it does not necessarily tracks genetic relationship. RFLPs have been shown to change in as few as 10 animal passages (3), and a single mutation (depending on where it occurs within ORF5) has the potential to change the RFLP type. This is referred to as "RFLP instability." Additionally, distantly related PRRS viruses regularly share the same RFLP type (see Supplementary Table 1 of Paploski 2021) (4). These factors make the immunological meaning of RFLP types unclear, partly because it is not always clear whether viruses that share the same RFLP type are homologous and whether viruses with different RFLP types are truly heterologous.
So how are RFLPs used in the daily routine of a farm? The first use is to help differentiate wild-type PRRS from strains utilized in some modified live vaccines. Some vaccines in the market have a particular RFLP pattern that is practically exclusive to the vaccine. This is not true to all vaccines commercially available.
Read the full story at 20220412NHF-001_553&sfvc4enews=42&cl=article_1&utm_rid=CPG02000003679044&utm_campaign=66753&utm_medium=email&elq2=2b1b3357e54542eb9303d64ca30488fe&sp_eh=&sp_eh=” target=”_blank”>National Hog Farmer 12 April 2022]